The selective action of several antibiotics on protein synthesis in bacteria will be further analyzed. We have found that streptomycin, spectinomycin, and erythromycin block initiating ribosomes at some step, but at different stages, shortly after formation of the initiation complex, and the blocks are unstable. We plan to identify these stages and to extend this approach to additional antibiotics. We will use purified initiation factors and elongation factors and specific antisera to control the step-by-step reaction. The possible role of a ribosome release factor in the release of ribosomes from such blocked initiating complexes will be analyzed. Though streptomycin has long been considered a model for the action of all aminoglycosides on ribosomes, it binds to only one site, and we have recently obtained evidence for a second-site binding of gentamicin that increases misreading but paradoxically decreases inhibition. We will expand the same approaches to other aminoglycosides, and we will isolate multiple-step resistant mutants, which will be used to study the interactions of the target sites. We have recently demonstrated a physiological function of membrane-bound polysome, which synthesize proteins that are simultaneously being secreted. We propose to study the suggested differential actions of antibiotics on these polysomes and on free polysomes. If a difference can be firmly established its mechanism will be further analyzed to determine whether it is due to the attached membrane or to a difference in the peptides being produced. The present studies should make it possible to pin-point the mechanisms of action of certain antibiotics in greater detail. In addition, as in all fundamental studies on antibiotic action, the knowledge gained will help to advance our understanding of the cell components that are affected: e.g., conformational differences between ribosomes blocked in different states by different antibiotics, and the functional interaction of membrane and ribosomes.